Fusion Proteins Having a Toxin and Cancer Marker, Nanoparticles, and Uses Related Thereto

ABSTRACT

This disclosure relates to nanoparticles coated with fusion proteins comprising a domain that binds a cancer marker and a domain comprising a toxic polypeptide. In certain embodiments, the targeted cancer marker is urokinase plasminogen activator receptor (uPAR) insulin-like growth factor 1 receptor (IGF1R), EGFR, HER2, and/or other member of the ErbB family of receptors. In certain embodiments, the molecule that binds a cancer marker is an amino terminal fragment of uPA or variant capable of binding uPAR and/or IGF1 or variant capable of binding IGF1R. In certain embodiments, the toxic polypeptide is a bacterial exotoxin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/651,699filed Mar. 27, 2020, which is the National Stage of InternationalApplication No. PCT/US2018/053156 filed Sep. 27, 2018, which claims thebenefit of U.S. Provisional Application No. 62/563,828 filed Sep. 27,2017. The entirety of each of these applications is hereby incorporatedby reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under CA154129,CA151810, and CA154846 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM

The Sequence Listing associated with this application is provided in XMLformat and is hereby incorporated by reference into the specification.The name of the XML file containing the Sequence Listing is15176USDIV.xml. The XML file is 15 KB, was created on Mar. 10, 2023, andis being submitted electronically via the USPTO patent electronic filingsystem.

BACKGROUND

Resistance to chemotherapy and radiotherapy is the major challenge incancer treatment that leads to cancer progress and a poor survival ofcancer patients. Many patients fail to respond to the initial treatmentof the combination therapy of several chemotherapy drugs (primaryresistance) whereas some patients initially respond favorably to aspecific therapeutic intervention then develop resistance over time(acquired resistance). Most likely, those tumors are not only resistantto the chemotherapeutic agents that were used to treat the patients, butalso respond poorly to other chemotherapy drugs. Thus, there is a needto identify improved cancer treatments.

Several reports explored the application of PE38 toxin for targetedcancer therapy. For example, Kreitman et al. report anti-CD22recombinant immunotoxin moxetumomab pasudotox. Clin Cancer Res. 2011,17(20):6398-405. Zielinski et al. report a recombinant, HER2-specific,affitoxin as an anticancer agent for targeted therapy of HER2-positivetumors. J Immunother. 2009, 32(8):817-25

Several reports explored the application of nanoparticles for targetedcancer therapy. For example, Bombelli et al. report nanoparticletherapies for future metastatic melanoma treatment. Lancet Oncol. 2014,15(1):e22-32. See also Zhou et al. IGF1 Receptor Targeted TheranosticNanoparticles for Targeted and Image-Guided Therapy of PancreaticCancer. ACS Nano, 2015, 9(8):7976-91; Yang et al. TheranosticNanoparticles Carrying Doxorubicin Attenuate Targeting Ligand SpecificAntibody Responses Following Systemic Delivery, Theranostics, 2015,5(1):43-61; Huang et al. Casein-coated Iron Oxide Nanoparticles for HighMM Contrast Enhancement and Efficient Cell Targeting, ACS appliedmaterials & interfaces, 2013, 5(11):4632-4639. Lee et al. TheranosticNanoparticles with Controlled Release of Gemcitabine for TargetedTherapy and MRI of Pancreatic Cancer, ACS nano, 2013, 7(3):2078-2089.See WO 2012/031205, WO 2013/0343996, CN103304638.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to nanoparticles coated with fusion proteincomprising a domain that binds a cancer marker and a domain comprising atoxic polypeptide. In certain embodiments, the targeted cancer marker isurokinase plasminogen activator receptor (uPAR) insulin-like growthfactor 1 receptor (IGF1R), EGFR, HER2, and/or other member of the ErbBfamily of receptors. In certain embodiments, the molecule that binds acancer marker is an amino terminal fragment of uPA or variant capable ofbinding uPAR and/or IGF1, variant capable of binding IGF1R, or peptidesor antibody fragment capable of binding EGFR. In certain embodiments,the toxic polypeptide is a bacterial exotoxin such as Pseudomonasexotoxin or fragment.

In certain embodiments the disclosure contemplates an ATF-uPA-spacer-PE38 fusion peptide comprising:

(SEQ ID NO: 1) NCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDELor variants thereof.

In certain embodiments the disclosure contemplates a ScFvEGFR-spacer-PE38 fusion peptide comprising:

(SEQ ID NO: 5) TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL

In certain embodiments, the disclosure relates the methods of usingfusion proteins or nanoparticles disclosed herein for the treatment ofcancer. In certain embodiments, the disclosure relates the methods ofusing nanoparticles disclosed herein for therapeutic and diagnosticapplications. In certain embodiments, the fusion protein comprises SEQID NO: 1, 3, 4, 5 or variants thereof.

In certain embodiments, the disclosure relates to targeted delivery ofnanoparticles into tumors mediated by fusion proteins disclosed herein.In certain embodiments, the disclosure contemplates nanoparticlescomprising fusion proteins having an amino terminal fragment of uPAcapable of binding uPAR wherein the nanoparticles further comprises IGF1as a dual uPAR and IGF1R targeted delivery system.

In certain embodiments, the disclosure contemplates targeted delivery offusion proteins disclosed herein, such as those comprising SEQ ID NO: 1,3, 4, 5 or variants, into tumors to reduce the growth of cancerouscells.

In certain embodiments, this disclosure relates to a nucleic acidsequence that encodes a fusion protein disclosed herein. In furtherembodiments, this disclosure relates to a vector comprising a nucleicacid sequence that encodes a fusion protein disclosed herein. In certainembodiments, this disclosure relates to a cell comprising a vectorcomprising a nucleic acid sequence that encodes a fusion proteindisclosed herein. In certain embodiments, this disclosure relates to anexpression system comprising a vector comprising a nucleic acid sequencethat encodes a fusion protein disclosed herein.

In certain embodiments, this disclosure relates to a fusion peptidevariant (SEQ ID NO: 1, 3, 4, or 5) wherein the variant has at least 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 98 percent sequence identityor similarity. In certain embodiments, the variant has one or more or upto 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions,and/or additions. In certain embodiments, the substitution is aconserved substitution.

In certain embodiments, this disclosure relates to a fusion proteinvariant of SEQ ID NO: or 3, that is capable of binding uPAR and reducingthe growth of cancer cells. In certain embodiments, this disclosurerelates to a fusion protein variant of SEQ ID NO: 4 or 5, that iscapable of binding EGFR and reducing the growth of cancer cells.

In certain embodiments, this disclosure relates to a nanoparticlecomprising a fusion protein disclosed herein wherein the nanoparticlecomprises 20 to 30 or 10 to 40 or 10 to 60 or 10 to 100 of the peptidemoieties bound to the exterior of the particle. In certain embodiments,this disclosure relates to a nanoparticle comprising a core comprisingiron oxide, gold, or silver as well as polymeric nanoparticles. Incertain embodiments, the core has an average diameter of 4 to 10, or 3to 20, or 3 to 50 nm, or 3 to 500 nm. In certain embodiments, themetallic nanoparticles core have an average diameter of 4 to 10, 4 to 20nm, or 100 to 300 nm. In certain embodiments, the polymericnanoparticles 100 to 200 nm, or 50 to 200, or 50 to 500 nm.

In certain embodiments, this disclosure relates to a pharmaceuticalcomposition comprising a nanoparticle disclosed herein and apharmaceutically acceptable excipient. In further embodiments, thisdisclosure relates to a pharmaceutical composition, in an aqueousphosphate buffer solution. In certain embodiments, this disclosurerelates to a pharmaceutical composition in the form of a pill, capsule,tablet, cream, or aerosol.

In certain embodiments, this disclosure relates to a method of treatingcancer comprising administering an effective amount of a fusion proteindisclosed herein or a nanoparticle comprising a fusion protein herein,e.g., a peptide comprising SEQ ID NO: 1, 3, 4, 5, or variants thereof,to a subject in need thereof. In certain embodiments, the cancer isselected from carcinoma, lymphoma, blastoma, sarcoma, leukemia,non-small cell lung, squamous cell, small-cell lung, peritoneum,hepatocellular, gastrointestinal, pancreatic, glioma, cervical, ovarian,liver, bladder, hepatoma, breast, colon, colorectal, endometrial,uterine, salivary gland, kidney, liver, prostate, vulval, thyroid,hepatic, and other lymphoproliferative disorders, and various types ofhead and neck. In certain embodiments, the cancer can be a primary ormetastatic tumors.

In certain embodiments, the nanoparticles or fusion proteins disclosedherein are administered intravenously or intraperitoneally.

In further embodiments, this disclosure relates to methods of treatingcancer further comprising administering a second nanoparticle and/orchemotherapy agent to the subject.

In certain embodiments, this disclosure relates to a method for cancerdiagnosis comprising administering an effective amount of a peptidedisclosed herein or nanoparticle disclosed herein to a subject in needthereof and detecting the particle about the area of a cancerous cell ortumor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a fusion protein disclosed herein (SEQ ID NO: 3) anda nucleic acid (SEQ ID NO: 2) encoding the fusion protein uPAR targetedhuman ATF (39)-PE38 Toxin.

FIG. 2 illustrates a fusion protein ScFvEGFR-PE38 toxin (SEQ ID NO: 4).

FIG. 3 illustrates conjugation of dual IGF1-IR and uPAR targeted,NIR-830 dye-IGF1 and ATF-Toxin to an amphiphilic polymer coated IONPs.

FIG. 4 shows data on in vitro cytotoxicity of unconjugated non-targetingPE38 (NT-toxin), ATF-PE38 (ATF-Toxin) and their related IONP conjugates.Preliminary breast cancer cells were derived from human breast PDXtumors.

FIG. 5 shows data on in vivo antitumor effect in an orthotopic humanbreast PDX tumor model. Growth inhibition of breast PDX tumors bydifferent treatments. Mice were i.v. administrated at a toxin dose of 2mg/kg twice a week for 5 injections.

FIG. 6 shows data on mean tumor weight of each treatment group aftersurgical collection of the tumors.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

The term “nanoparticle” refers to a molecular conglomerate of aboutbetween 1 and 1000 nm in diameter. One more molecules or biomoleculeslinked to the nanoparticle typically refers to covalently attaching themolecules or biomolecules to a polymer based exterior or coating.

Within certain embodiment, the compositions and methods disclosed hereinmay be utilized with a variety of polymer coated particle such as, e.g.,quantum dots (QDs), metal particles, gold, silver, iron, and iron-oxidenanoparticles (IONPs).

An “isolated” peptide refers one that its sequence was synthesizedchemically or by recombinant techniques and purified/isolated aftersynthesis. The peptide sequence is not purified from naturally occurringenvironment but may be derived from genetically modified cells or plantsor bacterial expression systems.

A “specific binding” refers to binding by molecules, such aspolynucleotides, antibodies, and other ligands, that are able to bind toor recognize a binding partner (or a limited number of binding partners)to a substantially higher degree than to other, similar biologicalentities.

A “subject” is defined to include any living animal or human. The term“non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc. A subject or non-human animal is “treated” ifone or more beneficial or desired results, including desirably clinicalresults, are obtained. For purposes of this disclosure, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: decreasing one or more symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, delaying the progression of the disease, and/or prolongingsurvival of individuals.

A “nucleic acid,” or “oligonucleotide,” is defined as a polymer ofnucleotides. As used herein, a “nucleotide” is given its ordinarymeaning as used in the art, i.e., a molecule comprising a sugar moiety,a phosphate group, and a base (usually nitrogenous). Typically, thenucleotide comprises one or more bases connected to a sugar-phosphatebackbone (a base connected only to a sugar moiety, without the phosphategroup, is a “nucleoside”). The sugars within the nucleotide can be, forexample, ribose sugars (a “ribonucleic acid,” or “RNA”), or deoxyribosesugars (a “deoxyribonucleic acid,” or “DNA”). In some cases, the polymercan comprise both ribose and deoxyribose sugars. Examples of basesinclude, but not limited to, the naturally-occurring bases (e.g.,adenosine or “A,” thymidine or “T,” guanosine or “G,” cytidine or “C,”or uridine or “U”). In some cases, the polymer can also comprisenucleoside analogs (e.g., aracytidine, inosine, isoguanosine,nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine,2-thiothymidine, 3-deaza-5-azacytidine, 2′-deoxyuridine, 3-nitorpyrrole,4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine,2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine,6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine,8-azaadenosine, 8-azidoadenosine, benzimidazole, N¹-methyladenosine,pyrrolo-pyrimidine, 2-amino-6-chloropurine, 3-methyl adenosine,5-propynylcytidine, 5-propynyluridine, 5-bromouridine, 5-fluorouridine,5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, etc.), chemically orbiologically modified bases (e.g., methylated bases), intercalatedbases, modified sugars (e.g., 2′-fluororibose, 2′-aminoribose,2′-azidoribose, 2′-O-methylribose, L-enantiomeric nucleosides arabinose,hexose, etc.), modified phosphate moieties (e.g., phosphorothioates or5′-N-phosphoramidite linkages), and/or other naturally and non-naturallyoccurring bases substitutable into the polymer, including substitutedand unsubstituted aromatic moieties. Other suitable base and/or polymermodifications are well-known to those of skill in the art. In somecases, the polynucleotide can include DNA, RNA, modified DNA, modifiedRNA, antisense oligonucleotides, expression plasmid systems,nucleotides, modified nucleotides, nucleosides, modified nucleosides,intact genes, or combinations thereof. Other examples of polynucleotidesinclude interfering RNA, natural or unnatural siRNAs, shRNAs, microRNAs,ribozymes, DNA plasmids, antisense oligonucleotides, randomizedoligonucleotides, or ribozymes. A nucleic acid sequence may be composedof DNA nucleotides, RNA nucleotides or a combination of both types andmay include natural nucleotides, chemically modified nucleotides andsynthetic nucleotides.

“Amino acid sequence” is defined as a sequence composed of any one ofthe 20 naturally appearing amino acids, amino acids which have beenchemically modified, or composed of synthetic amino acids. The terms“protein” and “peptide” refer to compounds comprising amino acids joinedvia peptide bonds and are used interchangeably. As used herein, where“amino acid sequence” is recited herein to refer to an amino acidsequence of a protein molecule. An “amino acid sequence” can be deducedfrom the nucleic acid sequence encoding the protein.

Sequence “identity” refers to the number of matching residues (expressedas a percentage) in a sequence alignment between two sequences of thealignment. As used herein, percentage identity of an alignment iscalculated using the number of identical positions divided by thegreater of the shortest sequence or the number of equivalent positionsexcluding overhangs wherein internal gaps are counted as an equivalentposition.

Percent “similarity” is used to quantify the similarity between twosequences of the alignment. This method is identical to determining theidentity except that certain amino acids do not have to be identical tohave a match. Amino acids are classified as matches if they are among agroup with similar properties according to the following amino acidgroups: Aromatic—F Y W; hydrophobic-A V I L; Charged positive: R K H;Charged negative—D E; Polar—S T N Q.

The terms “variant” when used in reference to a polypeptide refer to anamino acid sequence that differs by one or more amino acids fromanother, usually related polypeptide. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties. One type of conservative amino acidsubstitutions refers to the interchangeability of residues havingsimilar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. More rarely, a variant may have “non-conservative”changes (e.g., replacement of a glycine with a tryptophan). Similarminor variations may also include amino acid deletions or insertions (inother words, additions), or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological activity may be found using computer programs wellknown in the art, for example, DNAStar software. Variants can be testedin functional assays. Certain variants have less than 10%, andpreferably less than 5%, and still more preferably less than 2% changes(whether substitutions, deletions, and so on).

The term “recombinant” when made in reference to a nucleic acid moleculerefers to a nucleic acid molecule which is comprised of segments ofnucleic acid joined together by means of molecular biologicaltechniques. The term “recombinant” when made in reference to a proteinor a polypeptide refers to a protein molecule which is expressed using arecombinant nucleic acid molecule.

The terms “vector” or “expression vector” refer to a recombinant nucleicacid containing a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism or expression system, e.g.,cellular or cell-free. Nucleic acid sequences necessary for expressionin prokaryotes usually include a promoter, an operator (optional), and aribosome binding site, often along with other sequences. Eukaryoticcells are known to utilize promoters, enhancers, and termination andpolyadenylation signals.

Protein “expression systems” refer to in vivo and in vitro (cell free)systems. Systems for recombinant protein expression typically utilizecells transfecting with a DNA expression vector that contains thetemplate. The cells are cultured under conditions such that theytranslate the desired protein. Expressed proteins are extracted forsubsequent purification. In vivo protein expression systems usingprokaryotic and eukaryotic cells are well known. Also, some proteins arerecovered using denaturants and protein-refolding procedures. Commonexpression systems, for the expression of a protein coded for by foreignDNA carried by the vector and introduced to the host cell, include E.coli host cells and plasmid vectors, insect host cells and Baculovirusvectors, and mammalian host cells and vectors. Other examples of hostcells include, without limitation, prokaryotic cells (such as bacteria)and eukaryotic cells (such as yeast cells, mammalian cells, insectcells, plant cells, etc.). Specific examples include E. coli,Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Verocells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary orestablished mammalian cell cultures (e.g., produced from lymphoblasts,fibroblasts, embryonic cells, epithelial cells, nervous cells,adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cell (ATCCCRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which adihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) isdefective, rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662, hereinafterreferred to as “YB2/0 cell”), and the like.

In vitro (cell-free) protein expression systems typically usetranslation-compatible extracts of whole cells or compositions thatcontain components sufficient for transcription, translation andoptionally post-translational modifications such as RNA polymerase,regulatory protein factors, transcription factors, ribosomes, tRNAcofactors, amino acids and nucleotides. In the presence of an expressionvectors, these extracts and components can synthesize proteins ofinterest. Cell-free systems typically do not contain proteases andenable labeling of the protein with modified amino acids. Some cell freesystems incorporated encoded components for translation into theexpression vector. See, e.g., Shimizu et al., Cell-free translationreconstituted with purified components, 2001, Nat. Biotechnol., 19,751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): e141,both hereby incorporated by reference in their entirety.

In certain embodiments, the disclosure relates to recombinant peptidescomprising sequences disclosed herein or variants or fusions thereofwherein the amino terminal end or the carbon terminal end of the aminoacid sequence are optionally attached to a heterologous amino acidsequence, label, or reporter molecule.

The term “fusion” when used in reference to a polypeptide refers to achimeric protein containing a protein of interest joined to an exogenousprotein fragment (the fusion partner). The fusion partner may servevarious functions, including enhancement of solubility of thepolypeptide of interest, as well as providing an “affinity tag” to allowpurification of the recombinant fusion polypeptide from a host cell orfrom a supernatant or from both. If desired, the fusion partner may beremoved from the protein of interest after or during purification.

A “label” refers to a detectable compound or composition that isconjugated directly or indirectly to another molecule, such as anantibody or a protein, to facilitate detection of that molecule.Specific, non-limiting examples of labels include fluorescent tags,enzymatic linkages, and radioactive isotopes. In one example, a “labelreceptor” refers to incorporation of a heterologous polypeptide in thereceptor. A label includes the incorporation of a radiolabeled aminoacid or the covalent attachment of biotinyl moieties to a polypeptidethat can be detected by marked avidin (for example, streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionucleotides (such as ³⁵S or¹³¹I) fluorescent labels (such as fluorescein isothiocyanate (FITC),rhodamine, lanthanide phosphors), enzymatic labels (such as horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (such as a leucine zipperpair sequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), or magnetic agents, such as gadolinium chelates.In some embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance.

In certain embodiments, the disclosure relates to the recombinantvectors comprising a nucleic acid encoding a peptide disclosed herein orfusion protein thereof and optionally a selectable marker. A “selectablemarker” is a nucleic acid introduced into a recombinant vector thatencodes a polypeptide that confers a trait suitable for artificialselection or identification (report gene), e.g., beta-lactamase confersantibiotic resistance, which allows an organism expressingbeta-lactamase to survive in the presence antibiotic in a growth medium.Another example is thymidine kinase, which makes the host sensitive toganciclovir selection. It may be a screenable marker that allows one todistinguish between wanted and unwanted cells based on the presence orabsence of an expected color. For example, the lac-z-gene produces abeta-galactosidase enzyme which confers a blue color in the presence ofX-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). If recombinantinsertion inactivates the lac-z-gene, then the resulting colonies arecolorless. There may be one or more selectable markers, e.g., an enzymethat can complement to the inability of an expression organism tosynthesize a particular compound required for its growth (auxotrophic)and one able to convert a compound to another that is toxic for growth.URA3, an orotidine-5′ phosphate decarboxylase, is necessary for uracilbiosynthesis and can complement ura3 mutants that are auxotrophic foruracil. URA3 also converts 5-fluoroorotic acid into the toxic compound5-fluorouracil. Additional contemplated selectable markers include anygenes that impart antibacterial resistance or express a fluorescentprotein. Examples include, but are not limited to, the following genes:amp^(r), cam^(r), tet^(r), blasticidin^(r), neo^(r), hyg^(r), abx^(r),neomycin phosphotransferase type II gene (nptII), p-glucuronidase (gus),green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase(lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat),alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphosresistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase(xylA), arabitol dehydrogenase (atlD), UDP-glucose:galactose-1-phosphateuridyltransferasel (galT), feedback-insensitive a subunit ofanthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR),benzyladenine-N-3-glucuronide, E. coli threonine deaminase, glutamate1-semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO),salt-tolerance gene (rstB), ferredoxin-like protein (pflp),trehalose-6-P synthase gene (AtTPS1), lysine racemase (lyr),dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1(AtTSB1), dehalogenase (dhlA), mannose-6-phosphate reductase gene(M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase(dsdA).

In certain embodiments, the recombinant vector optionally comprises amammalian, human, insect, viral, bacterial, bacterial plasmid, yeastassociated origin of replication or gene such as a gene or retroviralgene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotidesegment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx orstructural genes selected from gag, pol, and env.

In certain embodiments, the recombinant vector optionally comprises agene vector element (nucleic acid) such as a selectable marker region,lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG)promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerasepromoter, SV40 promoter, internal ribosome entry site (IRES) sequence,cis-acting woodchuck post regulatory element (WPRE), scaffold-attachmentregion (SAR), inverted terminal repeats (ITR), FLAG tag coding region,c-myc tag coding region, metal affinity tag coding region, streptavidinbinding peptide tag coding region, polyHis tag coding region, HA tagcoding region, MBP tag coding region, GST tag coding region,polyadenylation coding region, SV40 polyadenylation signal, SV40 originof replication, Col E1 origin of replication, f1 origin, pBR322 origin,or pUC origin, TEV protease recognition site, loxP site, Cre recombinasecoding region, or a multiple cloning site such as having 5, 6, or 7 ormore restriction sites within a continuous segment of less than 50 or 60nucleotides or having 3 or 4 or more restriction sites with a continuoussegment of less than 20 or 30 nucleotides.

“Radiation therapy” is defined as a cancer treatment that useshigh-energy x-rays or other types of radiation to kill cancer cells orkeep them from growing. There are two types of radiation therapy.External radiation therapy, which uses a machine outside the body tosend radiation to the cancer. Internal radiation therapy uses aradioactive substance sealed in needles, seeds, wires, or catheters thatare placed directly into or near the cancer. The way the radiationtherapy is administer is directly dependent on the type and stage of thecancer.

“Chemoradiation therapy” is defined as a therapy that combineschemotherapy and radiation therapy to increase the effects of both.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” may be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample compound compared to a control without the compound. It may alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers. The cancer to be treated in the context of the presentdisclosure may be any type of cancer or tumor.

With regard to fusions proteins disclosed herein, a “toxin” refers to apolypeptide that impedes or slows the growth of a cancerous cell ortumor. PE38 is toxin which is a truncated portion of Pseudomonasaeruginosa exotoxin A (PE) having the following amino acid sequence

(SEQ ID NO: 11) EGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPG.In certain embodiments, the disclosure is not limited to the PE38 toxinas other toxic peptides are contemplated.

Nanoparticles

This disclosure relates to nanoparticles comprising a toxin or a fusionprotein that acts as a targeting moiety and a toxin. In certainembodiments, the fusion protein comprises SEQ ID NO: 1, 3, 4, 5, orvariants thereof. When reference is made to a metal particle ornanoparticle comprising a peptide or toxin, it is understood that thepeptide, fusion protein, and/or toxin is bound to the particle through apolymer coating, either through covalent bonds or other bindinginteractions, e.g., hydrophobic or hydrophilic binding or chelatinginteractions.

Within certain embodiment, the compositions and methods disclosed hereinmay be utilized with a variety of polymer coated particle such as, e.g.,quantum dots (QDs), metal particles, gold, silver, iron, and iron-oxidenanoparticles (IONPs). IONPs are typically prepared with a mean particlediameter of 4-100 nm. IONPs may be prepared by aging a stoichiometricmixture of ferrous and ferric salts in aqueous media under basicconditions. Control over particle size (2-20 nm) and shape is providedby adjusting the pH, ionic strength and the concentration of the growthsolution. The nanoparticles can be functionalized in situ usingadditives such as organic compounds (e.g. sodium citric) or polymers(e.g. dextran, polyvinyl alcohol). Other metals such as gold, cobalt,nickel, and manganese may be incorporated into the material.

High-temperature decomposition of Fe(CO)₅ in organic solvents is anotherway to prepare IONPs. Size (3-19 nm) can be varied using alternativetemperatures. Flame spray pyrolysis yields a range of magnetite,maghemite and wustite (FeO) particles IONPs. Iron precursor such asFe(CO)₅ and Fe(NO₃)₃ may be used. Flame spray pyrolysis can be used toproduce different nanoparticles (TiO₂, ZrO₂, silica, etc.) as well ashybrid particles (e.g. silica-IONPs).

Hydroxyl groups on the IONP provide a place for synthetic attachment ofdifferent functional groups. A range of chemistries can be used tostabilize metal nanoparticles, exploiting electrostatic, hydrophobic,chelating and covalent interactions. Carboxylic acid groups can interactwith the surface of IONPs by coordination processes. IONP synthesis inorganic solvents is typically conducted in oleic acid. A polymer coatingon the IONPs is preferred. Polymer attachment to the IONP surface by aninitiator fixed to the surface of the IONPs and the polymer is grownfrom the surface. Alternatively, a functional, pre-formed polymer isgrafted onto IONPs in situ. Copolymers with hydrophobic groups,carboxylic acid groups, polyethylene glycols, or amine groups arecontemplated. Polymers with a hydrophilic block and a hydrophobic blockare contemplated. See Yang et al., Clin Cancer Res, 2009 15:4722; Lin etal., Small, 2008, 4(3):334-341; Yu et a., Nanotechnology, 2006,17:4483-4487; Park et al., J. Mater. Chem., 2009, 19, 6412-6417; Boyeret al. NPG Asia Mater., 2010, 2(1):23-30, Kim et al., Nanotechnology,2011, 22, 155101; all hereby incorporated by reference in theirentirety.

Conjugating molecules or polypeptides to the polymers can beaccomplished using a variety of methods. Typically, primary aminecontaining compounds and proteins may be conjugated to the carboxylicacid groups on the polymer mediated by a coupling reagent such as EDAC.See Yang et al., Small, 2009, 5(2):235-43, hereby incorporated byreference in its entirety. Other coupling methods are contemplated,e.g., poly-histidine sequence may be recombinantly incorporated into apolypeptide sequence of the targeting moiety. A poly-histidine chelatingagent may be coupled to the polymer surface, e.g., NTA-Ni or NTA-Cu.Mixing the histidine tagged polypeptide sequence attaches it to thepolymer surface linked through the chelating agent NTA. Theavidin/streptavidin-biotin interactions may be used, e.g., biotin may becoupled to the polymer surface and streptavidin may be expressed as achimera with the targeting moiety.

In certain embodiments, a fusion protein disclosed herein comprises anamino-terminal fragment (ATF) of uPA, e.g., amino terminal fragment ofhuman uPA and a toxin.

In certain embodiments, the ATF-uPA comprises or consist of ATF,

135 aa (17 kDa) (SEQ ID NO: 6)SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGK.

In certain embodiments, the ATF-uPA comprises or consist of ATF,

68 aa of human uPA (SEQ ID NO: 7)SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTC YEGNGHFYRGKASTDTMG.

In certain embodiments, the ATF-uPA comprises or consist of ATF,

  39 (aa, 10-47) (SEQ ID NO: 8) NCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSK.

In the context of these sequences, term “comprise” refers to an ATF-uPAthat may contain more of the ATF-uPA sequence or other peptide, and theterm “consist of” refers to the sequence not having additional aminoacids on the N-terminal or C-terminal ends that match human ATF-uPA, butmay contain other peptide sequences.

In certain embodiments, the fusion protein disclosed herein comprises apeptide that binds EGFR.

ATF may be produced from E. coli BL21 bacterial expression system usinga pET20a plasmid (Invitrogen, Grand Island, N.Y.) containing the ATFcDNA sequence. Urokinase plasminogen activator (uPA) is a serineprotease that regulates multiple pathways involved in matrixdegradation, cell motility, metastasis and angiogenesis. Interaction ofthe N-terminal growth factor domain of uPA with its cellular receptor(uPAR) results in the conversion of the plasminogen to a serineprotease, which is a central regulator of the activation of otherproteases including the matrix metalloproteinases (MMPs). Studies haveshown that the uPA/uPAR complex controls the motility of both tumor andendothelial cells. In addition to its role in activation of the processfor degradation of extracellular matrix, uPAR also activates α5β1integrin and ERK signaling through interaction with EGFR and inducescell proliferation. Additionally, the uPA/uPAR complex can bind to thematrix protein, vitronectin, in association with transmembraneintegrins, and activate intracellular signaling molecules such as theprotein kinases, promoting cell adhesion, proliferation, and migration.

The uPAR-binding domain of uPA is located to the amino-terminal fragment(ATF) of uPA. Studies have shown that ATF is a potent uPA bindingantagonist to its high affinity receptor (uPAR) at the surface of bothtumor and endothelial cells. Systemic or local delivery of anon-catalytic amino-terminal fragment (ATF) of uPA (residues 1-135)using an adenoviral vector or conjugated peptides prevents the formationof the uPA/uPAR complex, thus inhibiting tumor growth and angiogenesis.Yang et al., Clin Cancer Res., 2009, 15(14):4722-32, hereby incorporatedby reference in its entirety, discuss the preparation of targeted ironoxide nanoparticle using a recombinant peptide containing theamino-terminal fragment of urokinase-type plasminogen activator (uPA)conjugated to magnetic iron oxide nanoparticles amino-terminal fragmentconjugated-iron oxide nanoparticle (ATF-IONP). This nanoparticle targetsuPA receptor, which is overexpressed in breast cancer tissues.

In certain embodiments, the peptide that binds EGFR may be

ScFVEGFR (SEQ ID NO: 9)TSEIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITF GQGTRLEIKRS.

In certain embodiments, nanoparticles disclosed herein comprise a fusionprotein disclosed herein that function as a targeting moiety and atoxin. In certain embodiments, the nanoparticles further comprise asecond targeting moiety such as human

IGF1 (SEQ ID NO: 10) MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKN YRM.

In certain embodiments, the second targeting moiety is a moiety thatbinds EGFR. The human epidermal growth factor receptor (EGFR) familyincludes EGFR (HER-1), EGFR-2 (HER-2), EGFR-3 (Her-3) and EGFR 4(HER-4). The ligands that bind to EGFRs are divided into EGFR-likeligands such as EGF and TGF-α, and the heregulins. These ligands bind toEGFR monomers to promoter receptor dimerization and oligomerization thatultimately results in the activation of the EGFR signaling pathway. ThisEGFR signaling pathway plays a role in the regulation of cellproliferation, survival and differentiation.

Human breast carcinomas express high levels of the EGF receptors.Overexpression of this receptor has been associated with highlyaggressive breast cancer types and a poor response to therapeuticagents. Prior preclinical and clinical studies have shown that blockingthe EGFR via monoclonal antibodies or inhibition of EGFR tyrosine kinasewith small molecule inhibitors inhibits the growth of breast cancers andsensitize chemotherapy responses. Single-chain antibodies to EGFR thatcontain the specific EGFR binding region but lack the Fc region havebeen isolated from human scFv phage display libraries. Yang et al.,Small, 2009, 5(2):235-43, hereby incorporated by reference in itsentirety, discuss the preparation of EGFR targeted nanoparticlesconjugating a single-chain anti-EGFR antibody (ScFvEGFR).

Iron oxide nanoparticles conjugated to a purified antibody thatselectively binds to the epidermal growth factor receptor (EGFR)deletion mutant (EGFRvIII) present on human glioblastoma multiforme(GBM) cells were used for therapeutic targeting and Mill contrastenhancement of experimental glioblastoma, both in vitro and in vivo,after convection-enhanced delivery (CED). See Hadjipanayis et al.,Cancer Res, 2010, 70:6303, hereby incorporated by reference in itsentirety. In certain embodiments, the disclosure relates to targetingmoiety that is an antibody or antibody mimetic to EGFR or EGFRvIII foruse in treating glioblastoma multiforme.

In certain embodiments, the second targeting moiety is a monoclonalantibody-610 that targets a surface antigen for use in treating coloncarcinoma. See Cerdan et al., Magn Reson Med, 1989, 12:151-63 1989,hereby incorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is an antibody tocarcinoembryonic antigen (CEA) that targets CEA for use in treatingcolon tumors. See Tiefenauer et al., Magn Reson Imaging, 1996,14:391-402, hereby incorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is a monoclonalantibody L6 that targets a surface antigen for use in treatingintracranial tumor. See Remsen et al., Am J Neuroradiol, 1996,17:411-18, hereby incorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is transferrin thattargets transferrin receptor for use in treating carcinoma. See Kresseet al., Magn Reson Med, 1998, 40:236-42, hereby incorporated byreference in its entirety.

In certain embodiments, the second targeting moiety is a monoclonalantibody to Her-2, e.g., Herceptin, which targets Her-2 receptors foruse in treating breast cancer. See Lee et al., Nat Med, 2007, 13:95-9;Artemov et al., Magn Reson Med, 2003, 49:403-8; and Huh et al., J AmChem Soc, 2005, 127:12387-91, all hereby incorporated by reference intheir entirety.

In certain embodiments, the second targeting moiety is the EPPT peptidethat targets underglycosylated mucin-1 antigen (uMUC-1) for use intreating breast, colon, pancreas, and lung cancer. See Moore et al.,Cancer Res, 2004, 64:1821-7, hereby incorporated by reference in itsentirety.

In certain embodiments, the second targeting moiety is folic acid thattargets folate receptor for use in treating mouth carcinoma and cervicalcancer. See Chen et al., PDA J Pharm Sci Technol, 2007, 61:303-13; Sunet al., Small, 2006, 4:372-9; and Sonvico et al., Bioconjug Chem, 2005,16:1181-8, all hereby incorporated by reference in their entirety.

In certain embodiments, the second targeting moiety is methotrexate thattargets folate receptor for use in treating cervical cancer. See Kohleret al., Langmuir, 2005, 21:8858-64, hereby incorporated by reference inits entirety.

In certain embodiments, the second targeting moiety is a monoclonalantibody A7 that targets colorectal tumor antigen for use in treatingcolorectal carcinoma. See Toma et al., Br J Cancer, 2005, 93:131-6,hereby incorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is chlorotoxinpeptide that targets membrane-bound matrixmetalloproteinase-2 (MMP-2)for use in treating glioma. See Veiseh et al., Nano Lett, 2005,5:1003-8, hereby incorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is F3 peptide thattargets surface-localized tumor vasculature for use in treating glioma.See Reddy et al., Clin Cancer Res, 2006, 12:6677-86, hereby incorporatedby reference in its entirety.

In certain embodiments, the second targeting moiety is RGD or RGD4C thattargets integrins for use in treating melanoma and epidermoid carcinoma.See Zhang et al., Cancer Res, 2007, 67:1555-62 and Uchida et al., J AmChem Soc, 2006, 128:16626-33, both hereby incorporated by reference intheir entirety.

In certain embodiments, the second targeting moiety is luteinizinghormone releasing hormone (LHRH) that targets LHRH receptor for use intreating breast cancer. See Leuschner et al., Breast Cancer Res Treat,2006, 99:163-76, hereby incorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is CREKA peptidethat targets clotted plasma proteins for use in treating breast cancer.See Simberg et al., Proc Natl Acad Sci USA, 2007, 104:932-6, herebyincorporated by reference in its entirety.

In certain embodiments, the second targeting moiety is an antibody toprostate specific membrane antigen (PSMA) that targets PSMA for use intreating prostate cancer. See Serda et al., Mol Imaging, 2007, 6:277-88,hereby incorporated by reference in its entirety.

In certain embodiments, the disclosure relates to multifunctionalnanoparticles comprising a targeting peptide disclosed herein, thenanoparticle, and a cargo. The nanoparticles can be either Quantum Dots(QDs) or gold nanoparticles that can be imaged optically or iron oxidenanoparticles (IONPs) that can be imaged via MRI. In certain embodimentsthe cargo is either a DNA cassette coding for a siRNA against anoncogene or survival factor, a chemotherapy drug or both.

Since siRNA is expressed from a RNA polymerase III (e.g., U6 or H1)promoter, a short hairpin siRNA (shRNA) gene may be cloned intoexpression vectors containing a polymerase III promoter to produceshRNAs from plasmid or viral vectors following transfecting into cells.See Brummelkamp et al., Science, 2002, 296, 550-553; Miyagishi & Taira,Nat. Biotechnol, 2002, 20, 497-500; McAnuff et al, J. Pharm. Sci. 2007,96, 2922-2930; Bot et al., Blood, 2005, 106, 1147-1153. The shRNAs arefurther processed into siRNAs by a cellular endoribonuclease. DNAcassettes expressing shRNA containing a U6 promoter and a shRNA gene canbe synthesized by a two-step PCR amplification protocol. See Castanottoet al., RNA, 2002, 8, 1454-1460 and Gou et al., FEBS Lett., 2003, 548,113-118.

In certain embodiments provided herein is a particle that contains apolymer-coated nanoparticle core, e.g., a fluorescent quantum dot (QD)or MRI contrast enhancing magnetic iron oxide nanoparticle (IONP),conjugated with about 10 to 20 DNA nanocassettes that contain a U6promoter and a shRNA gene for in vivo siRNA gene expression followingintracellular delivery. The nanoparticle is conjugated to a targetingpeptide disclosed herein typically the amino terminal fragment (ATF) ofthe urokinase plasminogen activator (uPA), which targets its cellularreceptor, uPAR. This receptor is highly expressed in tumors, angiogenicendothelial, and stromal cells in many types of human cancers. SeeNielsen et al., Int. J. Cancer 2007, 120, 2086-2095; Blasi & Carmeliet,Nat. Rev. Mol. Cell Biol. 2002, 3, 932-943; Pyke et al., Cancer Res,1993, 53, 1911-1915.

In certain embodiments, the disclosure relates to particles comprising acore coated with a polymer, wherein the polymer is conjugated to atargeting moiety, a lysosomally degradable moiety, and a therapeuticagent such as gemcitabine, doxorubicin, cytosine arabinoside, mitomycin,or any therapeutic agent with that an amine side group. In certainembodiments, the therapeutic agent is cisplatin, camptothecin orderivative such as 7-ethyl-10-hydroxycamptothecin (SN-38) or7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin(CPT-11). In certain embodiments, the particle is a metal nanoparticleor metal oxide nanoparticle, such as an iron oxide nanoparticle orelemental iron core nanoparticle with an oxide coat, or a quantum dot,e.g., those with a diameter of between about 5 to 200 nm or 10 to 100nm. In certain embodiments, the lysosomally degradable moiety is thepolypeptide GFLG (SEQ ID NO: 12) linked to the therapeutic agent. Incertain embodiments, the disclosure relates to compositions comprising apolymer conjugated to a targeting moiety, lysosomally degradable moiety,and a therapeutic agent which are described herein. In one example, thelysosomally degradable moiety linked to the therapeutic agent is of theformula:

or salts or derivatives thereof optionally substituted with one or moresubstituents. In certain embodiments, the polymer is an amphiphilicpolymer comprising a hydrophobic section further comprising ahydrophobic chemotherapeutic agent.

In certain embodiments, nanoparticles are coated with a siloxane polymersuch as PEG-b-AGE polymer. Li et al report PEG-b-AGE polymer coatedmagnetic nanoparticle probes with facile functionalization andanti-fouling properties for reducing non-specific uptake and improvingbiomarker targeting. J. Mater. Chem. B, 2015, 3, 3591-3603

In certain embodiments, the particle further comprises a fluorescentdye, e.g., a (3,3-dimethyl-indol-1-ium-1-yl)-N-alkylsulfonate dye orsalt thereof such as one of the formula:

or salts or derivatives thereof optionally substituted with one or moresubstituents wherein X is S or NH and n is 2 to 22 or n is 4 to 22. Incertain embodiments, the dye is conjugated to the free thiol group oncysteine or free amino group of the peptides or proteins.

Methods of Use

In certain embodiments, this disclosure relates to a method of treatingcancer comprising administering an effective amount of a nanoparticlecomprising a fusion protein disclosed herein or a fusion proteindisclosed herein, to a subject in need thereof.

In certain embodiments, this disclosure relates to a method of treatingcancer comprising administering an effective amount of a nanoparticlecomprising a fusion protein comprising SEQ ID NO: 1, 3, 4, 5, orvariants to a subject in need thereof.

In certain embodiments, this disclosure relates to a method of treatingcancer comprising administering an effective amount of a fusion proteincomprising SEQ ID NO: 1, 3, 4, 5, or variants to a subject in needthereof. In certain embodiments, the fusion protein may be administeredin combination with a nanoparticle comprising an amino-terminal fragment(ATF) of uPA or the fusion protein may be incorporated into thenanoparticle.

In certain embodiments, the disclosure contemplates a combinationchemotherapy comprising the administration of a first agent incombination with a second agent, wherein the first agent is ananoparticle comprising a fusion protein having SEQ ID NO: 1, 3, 4, 5 orvariant thereof, wherein the second agent is a nanoparticle comprising aan amino-terminal fragment (ATF) of uPA and a chemotherapy agentattached to the nanoparticle or the chemotherapy agent is encapsulatedby a polymer around the core of the particle.

In certain embodiments, the cancer overexpresses a receptor of atargeting molecule in tumor cells, tumor endothelial cells, or tumorstromal fibroblasts compared to noncancerous tissue of an organcontaining the cancerous tumor. In certain embodiments, the targetingmolecule is an antibody or fragment, antibody mimetic, inhibitor, oraptamer targeting a protein or glycoprotein expressed on the surface ofa cancerous cell. In certain embodiments, the cancer overexpress uPAR,IGF1R, EGFR, or HER-2. In certain embodiments, the cancer is selectedfrom pancreatic cancer, breast cancer, prostate cancer, lung cancer,skin cancer, bladder cancer, brain cancer, colon cancer, rectal cancer,kidney cancer, endometrial cancer, and thyroid cancer.

In certain embodiments, the cancer is selected from carcinoma, lymphoma,blastoma, sarcoma, and leukemia, non-small cell lung, squamous cell,small-cell lung, peritoneum, hepatocellular, gastrointestinal,pancreatic, glioma, cervical, ovarian, liver, bladder, hepatoma, breast,colon, colorectal, endometrial or uterine, salivary gland, kidney,liver, prostate, vulval, thyroid, hepatic, leukemia and otherlymphoproliferative disorders, and various types of head and neck. Incertain embodiments, the cancer can be a primary or metastatic tumors.

In further embodiments, this disclosure relates to methods of treatingcancer further comprising administering a particle disclosed hereincomprising a second chemotherapy agent or administering a secondchemotherapy to the subject separate from any chemotherapy agentcontained in or attached to the particle and/or the surrounding polymer.In certain embodiments, particles disclosed herein are administered aneffective amount to treat a subject diagnosed with cancer or a canceroustumor. In certain embodiments, the particles disclosed herein areadministered in combination with a second anti-cancer agent such as, butnot limited to, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1antibodies, nivolumab, pembrolizumab, ipilimumab, tremelimumab,bevacizumab, gefitinib, erlotinib, temozolomide, docetaxel, cisplatin,5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate,cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin,daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin,mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol,taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin,bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene,idoxifene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone,goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole,vorozole, exemestane, finasteride, marimastat, trastuzumab, cetuximab,dasatinib, imatinib, combretastatin, thalidomide, and/or lenalidomide orcombinations thereof.

In certain embodiments, the methods disclosed herein may be used incombination with radiation and chemoradiation therapy.

In certain embodiments, this disclosure relates to a method for cancerdiagnosis comprising administering an effective amount of a fusionprotein disclosed herein or nanoparticle disclosed herein to a subjectin need thereof and detecting the particle about the area of a cancerouscell or tumor.

Also contemplated are malignancies located in the colon, abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvis, skin, soft tissue, spleen, thorax and genito-urinaryapparatus and, more particularly, childhood acute lymphoblasticleukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia,acute myeloid leukemia, adrenocortical carcinoma, adult (primary)hepatocellular cancer, adult (primary) liver cancer, adult acutelymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin'sdisease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adultnon-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissuesarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, analcancer, astrocytoma, cancer of the biliary tract, cancer of the bladder,bone cancer, brain stem glioma, brain tumors, breast cancer, cancer ofthe renal pelvis and ureter, primary central nervous system lymphoma,central nervous system lymphoma, cerebellar astrocytoma, brainastrocytoma, cancer of the cervix, childhood (primary) hepatocellularcancer, childhood (primary) liver cancer, childhood acute lymphoblasticleukemia, childhood acute myeloid leukemia, childhood brain stem glioma,childhood cerebellar astrocytoma, childhood brain astrocytoma, childhoodextracranial germ cell tumors, childhood Hodgkin's disease, childhoodHodgkin's lymphoma, childhood visual pathway and hypothalamic glioma,childhood lymphoblastic leukemia, childhood medulloblastoma, childhoodnon-Hodgkin's lymphoma, childhood supratentorial primitiveneuroectodermal and pineal tumors, childhood primary liver cancer,childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhoodvisual pathway and hypothalamic glioma, chronic lymphocytic leukemia,chronic myeloid leukemia, cancer of the colon, cutaneous T-celllymphoma, endocrine pancreatic islet cells carcinoma, endometrialcancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing'ssarcoma and related tumors, cancer of the exocrine pancreas,extracranial germ cell tumor, extragonadal germ cell tumor, extrahepaticbiliary tract cancer, cancer of the eye, breast cancer in women,Gaucher's disease, cancer of the gallbladder, gastric cancer,gastrointestinal carcinoid tumor, gastrointestinal tumors, germ celltumors, gestational trophoblastic tumor, tricoleukemia, head and neckcancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma,hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers,intraocular melanoma, islet cell carcinoma, islet cell pancreaticcancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancerof the lip and mouth, cancer of the liver, cancer of the lung,lymphoproliferative disorders, macroglobulinemia, breast cancer in men,malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma,mesothelioma, occult primary metastatic squamous neck cancer, primarymetastatic squamous neck cancer, metastatic squamous neck cancer,multiple myeloma, multiple myeloma/plasmatic cell neoplasia,myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia,myeloproliferative disorders, paranasal sinus and nasal cavity cancer,nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma duringpregnancy, non-melanoma skin cancer, non-small cell lung cancer,metastatic squamous neck cancer with occult primary, buccopharyngealcancer, malignant fibrous histiocytoma, malignant fibrousosteosarcoma/histiocytoma of the bone, epithelial ovarian cancer,ovarian germ cell tumor, ovarian low malignant potential tumor,pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancerof the penis, pheochromocytoma, hypophysis tumor, neoplasia of plasmaticcells/multiple myeloma, primary central nervous system lymphoma, primaryliver cancer, prostate cancer, rectal cancer, renal cell cancer, cancerof the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancerof the salivary glands, sarcoidosis, sarcomas, skin cancer, small celllung cancer, small intestine cancer, soft tissue sarcoma, squamous neckcancer, stomach cancer, pineal and supratentorial primitiveneuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma,thyroid cancer, transitional cell cancer of the renal pelvis and ureter,transitional renal pelvis and ureter cancer, trophoblastic tumors, cellcancer of the renal pelvis and ureter, cancer of the urethra, cancer ofthe uterus, uterine sarcoma, vaginal cancer, optic pathway andhypothalamic glioma, cancer of the vulva, Waldenstrom'smacroglobulinemia, Wilms' tumor and any other hyperproliferativedisease, as well as neoplasia, located in the system of a previouslymentioned organ.

A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent” or thelike, refer to molecules that are recognized to aid in the treatment ofa cancer. Contemplated examples include the following molecules orderivatives such as temozolomide, carmustine, bevacizumab, procarbazine,lomustine, vincristine, gefitinib, erlotinib, cisplatin, carboplatin,oxaliplatin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed,methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin, mithramycin, vinblastine, vindesine, vinorelbine,paclitaxel, taxol, docetaxel, etoposide, teniposide, amsacrine,topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene,raloxifene, droloxifene, idoxifene, fulvestrant, bicalutamide,flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin,megestrol, anastrozole, letrozole, vorozole, exemestane, finasteride,marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin,thalidomide, azacitidine, azathioprine, capecitabine, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, doxifluridine, epothilone,irinotecan, mechlorethamine, mercaptopurine, mitoxantrone, pemetrexed,tioguanine, valrubicin and/or lenalidomide or combinations thereof suchas, cyclophosphamide, methotrexate, 5-fluorouracil (CMF); doxorubicin,cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone(MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD);cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP);bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin,5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX);methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

In certain embodiments, the disclosure relates to methods of optical andMRI imaging the nanoparticle in tumors. 3D-MRI enables monitoring ofintratumoral distribution of nanoparticles and tumor responses totherapeutics contained on or in the nanoparticles.

In certain embodiments, the disclosure relates to nanoparticles coatedwith amphiphilic polymers or PEG-b-AGE polymers conjugated withmolecules useful for targeting tumors, monitoring the location of thenanoparticles administered to a subject by MRI, and viewing the presenceof the nanoparticles during optical image-guided surgery.

In certain embodiments, the disclosure relates to uses of particlesdisclosed herein as a theranostics. Theranostics are therapeutics withphysical properties that allows one to image molecular accumulation ofthe vehicles in vivo. Yang et al., WO/2007/018647, disclose binding andinternalization of tumor targeted-iron oxide particles using MRI. Seealso Yang et al., J. Biomed. Nanotechnol., 2008, 4, 439-449. Lammers etal., Biomaterials, 2009, 30(2):3466-3475, disclose the simultaneousdelivery of doxorubicin and gemcitabine to tumors in vivo usingpolymeric drug carriers.

In certain embodiments, the disclosure relates to methods comprisingpreoperatively administering a composition comprising nanoparticlesdisclosed herein and monitoring the location of the particles in thesubject by detecting it by MRI (magnetic resonance imaging) in an areaof the subject. In certain embodiments, the method further comprises thesteps of operating on the subject in the area of detected particles,imaging dye identified tumors binding the targeting molecule, andsurgically removing dye identified tumors or tissue.

In certain embodiments, the disclosure relates to methods comprisingpreoperatively administering cancer targeted nanoparticles conjugated todyes disclosed herein to a subject, optically imaging a tumor that bindthe nanoparticles intra-operatively, and removing tumors targeted withthe nanoparticles.

In certain embodiments, the disclosure contemplates imaging andeffecting cancer cell lysis or other cell lysis with particles usingiron or iron oxide cores. See WO2009/120702.

In certain embodiments, the disclosure relates to targeting of cancer bylocal hyperthermia using composition and methods disclosed herein. Localhyperthermia can lead to induction of apoptosis, heat-shock proteinrelease, and chemotherapy agent sensitivity of cancer cells by exposureof cancer cells containing particles with an iron or iron oxide core toan alternating magnetic fields (<1000 kHz) that are safe to normalcells.

In certain embodiments, the disclosure relates to methods for lysis of acancer cells comprising, administering to a subject particles disclosedherein and adjusting magnetic fields proximate the subject to cause celllysis of cancer cell that absorb the particles after administration.Typically, the magnetic field is an oscillating magnetic field and theparticles are heated to at least 37° C. in vivo typically greater than41° C.

Pharmaceutical Compositions

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising particles disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thecomposition is a pill or in a capsule or the composition is an aqueousbuffer, e.g., a pH between 6 and 8. In certain embodiments, thepharmaceutically acceptable excipient is selected from a filler,glidant, binder, disintegrant, lubricant, and saccharide. Optionally,the pharmaceutical composition further comprises a second anticanceragent.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable (such as olive oil, sesame oil) and injectable organic esterssuch as ethyl oleate.

Prevention of the action of microorganisms may be controlled by additionof any of various antibacterial and antifungal agents, example,parabens, chlorobutanol, phenol, sorbic acid, and the like. It may alsobe desirable to include isotonic agents, for example sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the particlesmay be admixed with at least one inert customary excipient (or carrier)such as sodium citrate or dicalcium phosphate or: (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose, mannitoland silicic acid, (b) binders, as for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c)humectants, as for example, glycerol (d) disintegrating agents, as forexample, agar-agar, calcium carbonate, potato or tapioca starch, alginicacid, certain complex silicates, and sodium carbonate, (e) solutionretarders, as for example paraffin, (f) absorption accelerators, as forexample, quaternary ammonium compounds, (g) wetting agents, as forexample cetyl alcohol, and glycerol monostearate, (h) adsorbents, as forexample, kaolin and bentonite, and (i) lubricants, as for example, talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, or mixtures thereof. In the case of capsules, tablets,and pills, the dosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar and as high molecular weight polyethylene glycols, and thelike.

Solid dosage forms such as tablets, capsules, pills, and granules can beprepared with coatings and shells, such as enteric coatings and otherswell known in the art. They may contain opacifying agents, and can alsobe of such composition that they release the particles in a certain partof the intestinal tract in a delayed manner. Examples of embeddingcompositions which can be used are polymeric substances and waxes.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the particles, the liquid dosage forms may contain inertdiluents commonly used in the art, such as water or other solvents,solubilizing agents and emulsifiers, for example, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, viscoleo, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Suspensions, in addition to theparticles, may contain suspending agents, as for example, ethoxylatediso-stearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite agar-agarand tragacanth, or mixtures of these substances, and the like.

Pharmaceutical compositions typically comprise an effective amount ofparticles and a suitable pharmaceutical acceptable carrier. Thepreparations can be prepared in a manner known per se, which usuallyinvolves mixing the particles according to the disclosure with the oneor more pharmaceutically acceptable carriers, and, if desired, incombination with other pharmaceutical active compounds, when necessaryunder aseptic conditions. Reference is made to U.S. Pat. Nos. 6,372,778,6,369,086, 6,369,087 and 6,372,733 and the further references mentionedabove, as well as to the standard handbooks, such as the latest editionof Remington's Pharmaceutical Sciences.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and can be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which can be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe particles of the disclosure e.g., about 10, 25, 50, 100, 200, 300 or400 mg per unit dosage.

The particles can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The particles will generally be administered in an“effective amount,” by which it is meant any amount of particles that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered. Usually, depending on the condition to be prevented ortreated and the route of administration, such an effective amount willusually be between 0.01 to 1000 mg per kilogram body weight of thesubject per day, more often between 0.1 and 500 mg, such as between 1and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg,per kilogram body weight of the subject per day, which can beadministered as a single daily dose, divided over one or more dailydoses. The amount(s) to be administered, the route of administration andthe further treatment regimen can be determined by the treatingclinician, depending on factors such as the age, gender and generalcondition of the subject and the nature and severity of thedisease/symptoms to be treated.

Formulations containing particles described herein can be prepared usinga pharmaceutically acceptable carrier composed of materials that areconsidered safe and effective and can be administered to an individualwithout causing undesirable biological side effects or unwantedinteractions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Carrier also includes all components of the coating composition whichcan include plasticizers, pigments, colorants, stabilizing agents, andglidants. Delayed release, extended release, and/or pulsatile releasedosage formulations can be prepared as described in standard referencessuch as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (NewYork, Marcel Dekker, Inc., 1989), “Remington—The science and practice ofpharmacy,” 20th ed., Lippincott Williams & Wilkins, Baltimore, Md.,2000, and “Pharmaceutical dosage forms and drug delivery systems,” 6thEdition, Ansel et al., (Media, Pa: Williams and Wilkins, 1995). Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Diluents, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, gums or cross linked polymers,such as cross-linked PVP (Polyplasdone XL from GAF Chemical Corp).

Stabilizers are used to inhibit or retard decomposition reactions whichinclude, by way of example, oxidative reactions.

A “pharmaceutical composition” or “pharmaceutically acceptable”composition, is defined as a therapeutically effective amount of one ormore of the compositions described herein, formulated together with oneor more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail, the pharmaceutical compositions of thepresent disclosure can be specially formulated for administration insolid or liquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent material,involved in carrying or transporting the subject compound from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The compositions of the present disclosure can be given in dosages,generally, at the maximum amount while avoiding or minimizing anypotentially detrimental side effects. The compositions can beadministered in effective amounts, alone or in a cocktail with othercompounds, for example, other compounds that can be used to treat adisease. An effective amount is generally an amount sufficient toinhibit the disease within the subject.

One of skill in the art can determine what an effective amount of thecomposition is by screening the composition using known methods. Theeffective amounts may depend, of course, on factors such as the severityof the condition being treated; individual patient parameters includingage, physical condition, size, and weight; concurrent treatments; thefrequency of treatment; or the mode of administration. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. In some cases, a maximum dosebe used, that is, the highest safe dose according to sound medicaljudgment.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this disclosure can be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level may depend upon a variety of factors includingthe activity of the particular particles of the present disclosureemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular particles being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular particles employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the particles of the disclosure employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In some embodiments, particles or pharmaceutical composition of thedisclosure is provided to a subject chronically. Chronic treatmentsinclude any form of repeated administration for an extended period oftime, such as repeated administrations for one or more months, between amonth and a year, one or more years, or longer. In many embodiments, achronic treatment involves administering particles or pharmaceuticalcomposition of the disclosure repeatedly over the life of the subject.For example, chronic treatments can involve regular administrations, forexample one or more times a day, one or more times a week, or one ormore times a month. In general, a suitable dose such as a daily dose ofparticles of the disclosure will be that amount of the particles that isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.

While it is possible for a composition of the present disclosure to beadministered alone, it can be administered as a pharmaceuticalformulation (composition) as described above.

EXAMPLES Preparation and Purification of ATF-Toxin Fusion Protein:

A Pseudomonas exotoxin A (PE38KDEL) has a potent anti-tumor effect. Uponentering into cells, highly toxic PE38 domain interacts with thetranslation elongation factor 2 to inhibit protein synthesis, leading toapoptotic cell death. This cell death is significantly different fromall chemotherapy drugs that act through cellular DNA damage or blockingcell cycling. Although PE38 and other toxins have been evaluated ascancer therapeutic agents either by conjugating to antibodies or asfusion proteins, the major obstacle for clinical development is thepresence severe systemic toxicity due to nonspecific distribution intonormal tissues and organs. For recombination fusion protein mediatedtherapy, an additional issue is the short blood half-life that preventsefficient delivery into tumors. To address above issues, a tumortargeting ligand-toxin fusion proteins with was developed with dualreceptor targeting and cell death-inducing function. The targeting-toxinligand may conjugated to nanoparticles to be targeted delivered intotumor cells expressing those receptors, such as urokinase plasminogenactivator receptor (uPAR) and epidermal growth factor receptor (EGFR).Nanoparticle-mediated delivery of PE38 toxin significantly reducedsystemic toxicity while producing a strong antitumor growth effect.

Recombinant ATF-Toxin (PE38) was produced from a bacterial expressionsystem using a ATF-Toxin fusion gene plasmid. For production ofATF-Toxin, a frozen vial of about 1 mL of bacterial stock containingATF-Toxin plasmids was added into 50 mL of LB culture media (2.5% LBBroth media, 1% glucose and 100 ppb Ampicillin) and incubated overnightat 37° C. On the next day, the bacterial was transferred a 4 L flaskwith 1000 mL of LB culture media and cultured for about 3 h at 37° C.until the OD reading reaches 1.5˜1.8. 100 μL of IPTG (1 μM) was thenadded and the bacterial was cultured for 2 h. The flask was put into icebath for around half an hour and the bacterial was collected bycentrifugation at 6000 rpm for 5 min. The collected bacterial was storedat −20° C.

For purification of ATF-Toxin, the bacterial was dissolved in 30 mL oflysis buffer (50 mM NaH2PO4, 1 M NaCl and 0.4 M Urea). Imidazole (10 mM)and appropriate amount of protease inhibitor were added into the buffer.The bacterial was sonicated for 30 min in ice bath and debris wasremoved by centrifugation at 18000 rpm for 30 min. The supernatant wascollected and mixed with 1 mL of NTA-Ni-agarose beads (Qiagen, Miami,Fla.) for about 1 h at room temperature. The beads were collected bycentrifugation at 2000 rpm for 5 min and were transferred into apurification column (Poly-Prep Chromatography Columns, BioRad, Hercules,Calif.). The column was firstly eluted with 30 mL of washing buffer (50mM NaH2PO4, 1 M NaCl and 20 mM imidazole) to remove impurities and thenwith 2-5 mL of elution buffer (50 mM NaH2PO4, 0.3 M NaCl and 300 mMimidazole).

Production of uPAR Targeted ATF-Toxin or Dual uAPR and IGF1R TargetedIGF1-ATF-Toxin-IONP

Carboxyl groups on the amphiphilic polymer-coated IONPs (core size: 10nm) were activated by mixing with ethyl-3-dimethyl amino propylcarbodiimide (EDAC, Sigma-Aldrich) and N-hydroxysulfosuccinimide(sulfo-NHS, Sigma-Aldrich). NIR 830 dye-labeled fusion protein ofATF-PE38 (ATF-Toxin-Dye) alone or NIR 830 dye-labeled human recombinantIGF1 peptide (IGF1-Dye) in combination with NIR 830 dye-labeled fusionprotein of ATF-PE38 (ATF-Toxin-Dye) were conjugated to the polymercoated IONPs mediated by an amid bond between carboxyl groups on thesurface of the polymer and amine groups of IGF1 peptides or ATF-PE38peptide (FIG. 3 ). Although interaction of ATF with uPAR on the cellsurface can lead to endocytosis of ATF-Toxin, the level of uPARexpression is heterogeneous with a high level in the invasive tumor edgein both tumor cells and tumor stromal cells, and relatively low level inthe tumor center. However, IGF-1R is highly expressed in all tumorcells. The combination of uPAR and IGF-1R targeting increases targeteddelivery into tumor cells, especially in the tumor center.

In one example, 60 μg of EDAC and 40 μg of sulfo-NHS were added to theaqueous solution of 1 mg of IONPs in 10 mM borate buffer (pH 4.5),allowing activation of IONPs for 10 min. Activated IONPs were thencollected by centrifugation at 3000 rpm for 10 min using Nanosep 100 Kcolumn (Pall Corporation, Port Washington, N.Y., USA) and then reactedwith mixture of 75 μg of IGF1-Dye and 100 ug of ATF-Toxin-Dye in 10 mMborate buffer (pH 8.5) to produce ATF-Toxin-IONP or IGF1-ATF-Toxin-IONP.The reaction was carried out overnight (˜12 h) at 4° C. or for 4 h atroom temperature. The final ATF-Toxin-IONP or IGF1-ATF-Toxin-IONPs werepurified using Nanosep 100 K column. Control non-targeting Toxinconjugated IONPs (NT-Toxin-IONPs) were prepared using a PE38 proteinwithout targeting domain accordingly using same method.

In Vitro Cell Proliferation Assay

Human breast cancer cells derived from breast PDX tumors were culturedin 96-well plate at a density of 5,000 cells/well. After 24 h ofplating, cells were washed once with cold PBS. Different Toxin-IONPconjugates were diluted in the culture medium at various toxinconcentrations and then added into cell cultures. Unconjugated toxinswere used as a treatment control. After incubating for 4 h whenToxin-IONP conjugates bound to and entered into tumor cells, cells werewashed 3 times with cold PBS to remove unbound IONPs. 100 μL of freshmedium was then added to the plate. Cells were incubated for anadditional 72 h and viability of the cells was determined by Alamar Blueassay (Life Technologies, NY). Cells treated with culture medium alonewere used as the no treatment control. Results shown in FIG. 4 are themean value of four repeat studies.

In Vivo Near-Infrared Fluorescence Optical Imaging

Mice were subjected to NIR optical imaging when tumor reached a size ofabout 6-8 mm in diameter. 400 picomolar (pmol) of IGF1-IONPs,ATF-Toxin-IONPs and IGF1-ATF-Toxin-IONPs were injected via the tail veininto the tumor bearing mice. NIR optical imaging was conducted using theXenogen IVIS Spectrum imaging system (Caliper LIfe Sciences, Alameda,Calif.). Mice were sacrificed and tumors and normal organs werecollected. All optical images were captured using 745 nm excitation and820 nm emission filter set with 30 seconds exposure time. Optical Imageswere analyzed using the software provided by the Xenogen IVIS Spectrumimaging system. ROIs were selected for measuring the mean fluorescenceintensity (MFI) of tumors and corresponding body background. Micereceiving IGF1-IONPs, ATF-Toxin-IONPs and IGF1-ATF-Toxin-IONPs allshowed good IONP accumulation in the tumor sites.

In Vivo Antitumor Efficacy

Nude mice bearing orthotopic breast PDX tumors derived from the breastcancer Patient #9 tumor tissue (ER+, chemotherapy and Radiationresistant) were randomized into 4 experimental groups with 5 mice ineach group, including no treatment control, NT-Toxin-IONPs,ATF-Toxin-IONP, and IGF1-ATF-Toxin-IONP. Treatment started when thetumor reached sizes of 6-8 mm at an ATF-toxin equivalent dose of 2 mg/kgbody weight via the tail vein injection twice per week for 5 injections.Tumor volume and mouse body weight were monitored twice per week. Someof the tumors in the mice treated with targeted ATF-Toxin-IONP andIGF-ATF-Toxin-IONP turned to black color after 1st injection, suggestinginduction of tumor necrosis. All tumors became black in those groupafter 4th treatment. However, this was not found in NT-Toxin-IONPtreatment group. The tumors only became black when they reach certainsize (over 1.5 cm diameter).

After each injection, a representative mouse from each group wasselected for NIR optical imaging. The interesting thing for the micereceiving ATF-Toxin-IONP and IGF-ATF-Toxin-IONP is the tumors which hadoptical signals after 1st injection lose the optical signal aftermultiple treatment, while new grown tumor showed optical signal at thesame time. This confirmed the induction of tumor necrosis for thosegroups.

Tumors in all groups were collected by surgery 3 days after the finaltreatment and tumors were collected for histological, chemical, andimmunofluorescence analyses. In comparison with the no treatment controlgroup, the volumes of PDX tumors from the mice in all treated groupsshowed various degrees of tumor growth inhibition. We found thattreatment with NT-Toxin-IONP and ATF-Toxin-IONP led to 40.2% and 49.2%of tumor growth inhibition, respectively, compared to the control group(FIG. 5 ). However, there was no statistically significant differencebetween those two groups with no treatment control (Student's t-test: Notreatment control vs NT-Toxin-IONP: p=0.113, No treatment control vsATF-Toxin-IONP: p=0.088). In contrast, PDX tumors collected from themice that treated with IGF1-ATF-Toxin-IONP were significantly smallerthan those from the control groups of no treatment, or NT-Toxin-IONPtreated group. There was 77.7% tumor volume growth inhibition in theIGF1-ATF-Toxin-IONP treated mouse group. Differences in tumor volumesamong groups with different treatments were statistically significant(Student's t-test: No treatment control vs IGF1-ATF-Toxin-IONP p<0.03;NT-Toxin-IONP vs IGF1-ATF-Toxin-IONP p<0.02). Using loss of mouse bodyweight as an indication of system toxicity, there was no apparentsystemic toxicity for all treatment groups following 5 treatments of 2mg/kg of toxin equivalent doses.

In terms of tumor weights, similar results were found. Treatment withNT-Toxin-IONP, ATF-Toxin-IONP and IGF1-ATF-Toxin-IONP led to 43.3%,72.1% and 84.3% of tumor growth inhibition, respectively, compared tothe control group (FIG. 6 ). Statistically significant differences werefound between both ATF-Toxin-IONP and IGF1-ATF-Toxin-IONP with notreatment control.

1. A nanoparticle coated with a fusion peptide comprising a first domainhaving a single-chain anti-EGFR antibody sequence and the second domainof a toxin, and wherein the nanoparticle is further coated with amolecule that binds insulin-like growth factor 1 receptor.
 2. Thenanoparticle of claim 1 wherein the fusion peptide comprises the aminoacid sequence (SEQ ID NO: 5)TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL

or variants thereof, wherein the variant has at least 95% sequenceidentity.
 3. The nanoparticle of claim 1 wherein the fusion peptidecomprises the amino acid sequence (SEQ ID NO: 5)TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL.


4. The nanoparticle of claim 1, further comprising a chemotherapy agent.5. The nanoparticle of claim 4, wherein the chemotherapy agent isattached to a polymer around the core of the nanoparticle.
 6. Thenanoparticle of claim 4, wherein the chemotherapy agent is encapsulatedby a polymer around the core of the particle.
 7. The nanoparticle ofclaim 4, wherein the chemotherapy agent is doxorubicin.
 8. Thenanoparticle of claim 4, wherein the chemotherapy agent is cisplatin. 9.A pharmaceutical composition comprising a nanoparticle of claim 1 and apharmaceutically acceptable excipient.
 10. A method of treating cancercomprising administering an effective amount of a nanoparticlecomprising a chemotherapy agent to a subject in need thereof, whereinthe nanoparticle is coated with a fusion peptide comprising a firstdomain having a single-chain anti-EGFR antibody sequence and the seconddomain of a toxin, and wherein the nanoparticle is further coated with amolecule that binds insulin-like growth factor 1 receptor.
 11. Themethod of claim 10 wherein the fusion peptide comprises the amino acidsequence (SEQ ID NO: 5)TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL

or variants thereof, wherein the variant has at least 95% sequenceidentity.
 12. The method of claim 10 wherein the fusion peptidecomprises the amino acid sequence (SEQ ID NO: 5)TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL.


13. The method of claim 10, wherein the cancer is carcinoma, lymphoma,blastoma, sarcoma, leukemia, non-small cell lung cancer, squamous cellcancer, small-cell lung cancer, peritoneum cancer, hepatocellularcancer, gastrointestinal cancer, pancreatic cancer, glioma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial cancer, uterine,salivary gland cancer, kidney cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic cancer, head cancer, or neckcancer.
 14. A nucleic acid sequence comprising a sequence encoding thefusion protein having (SEQ ID NO: 5)TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL

or variants thereof, wherein the variant has at least 95% sequenceidentity.
 15. The nucleic acid of claim 14 wherein the fusion peptidecomprises the amino acid sequence (SEQ ID NO: 5)TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRSSGGGGSGAPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQ PGKPPKDEL.


16. A vector comprising a nucleic acid sequence of claim
 14. 17. A cellor cell free expression system comprising a vector of claim 14.